Centriolar Subdistal Appendages Promote Double-strand Break Repair Through Homologous Recombination

Article Centriolar subdistal appendages promote double-strand break repair through homologous recombination Guillermo Rodr ıguez-Real 1,2 , Andr es Dom ınguez-Calvo 1,2 , Rosario Prados-Carvajal 1,2 , Aleix Bayona-Feli u 3 , Sonia Gomes-Pereira 4 , Fernando R Balestra 1,2,* & Pablo Huertas1,2,** Abstract The centrosome is a cytoplasmic organelle with roles in microtubule organization that has also been proposed to act as a hub for cellular signaling. Some centrosomal components are required for full activation of the DNA damage response. However, whether the centrosome regulates specific DNA repair pathways is not known. Here, we show that centrosome presence is required to fully activate recombination, specifically to completely license its initial step, the so-called DNA end resection. Furthermore, we identify a centriolar structure, the subdistal appendages, and a specific factor, CEP170, as the critical centrosomal component involved in the regulation of recombination and resection. Cells lacking centrosomes or depleted for CEP170 are, consequently, hypersensitive to DNA damaging agents. Moreover, low levels of CEP170 in multiple cancer types correlate with an increase of the mutation burden associated with specific mutational signatures and a better prognosis, suggesting that changes in CEP170 can act as a mutation driver but could also be targeted to improve current oncological treatments. Keywords centrosomes; CEP170; DNA end resection; homologous recombination; subdistal appendages Subject Categories Cancer; Cell Adhesion, Polarity & Cytoskeleton; DNA Replication, Recombination & Repair DOI 10.15252/embr.202256724 | Received 23 December 2022 | Revised 18 July 2023 | Accepted 19 August 2023 EMBO Reports (2023)e56724 Introduction The centrosome is considered the major microtubule (MT) organizing center (MTOC) of animal cells, involved in mitotic spindle assembly, interphase MT-network organization and cilia and flagella assembly. Centrosomal defects, either in number or in structure, are linked to chromosome missegregation, polarity defects and motility or signaling defects related to the lack of cilia or flagella (Nigg & Raff, 2009; Bornens, 2012). The centrosome is composed of a pair of centrioles surrounded by a structured cloud of proteins known as the pericentriolar material (PCM) (Le Guennec et al,2021). The centriolar structure and its assembly pathway has been conserved throughout evolution (Nabais et al,2020). In humans, they are composed by nine triplets of MTs distributed in a radial symmetry to form a cylinder of approximately 500 nm long and 200 nm wide. In addition to MTs, from 200 to 300 proteins have been estimated to localize to the centrosome (Andersen et al,2003; Jakobsen et al,2011). However, only a minority of them has been described to play a role at the centrosome. For instance, the cartwheel component HsSAS-6 or the kinase PLK4 are required for centriolar duplication, thus the inactivation or depletion of either protein in human cultured cells leads to the inhibition of centriole assembly, rendering the appearance of cells without centrioles as the subsequent divisions go by (Wang et al,2015; Wong et al,2015;Go ¨nczy & Hatzopoulos, 2019). Interestingly, centriole-depleted cells can assemble a mitotic spindle and perform mitosis, although with a high rate of chromosome missegregation (Wang et al,2015; Wong et al,2015). These cells without centrioles expend a significant longer time in mitosis which leads to the activation of a p53-dependent pathway that leads to a G1 cell cycle arrest (Lambrus et al,2015,2016; Wong et al,2015; Fong et al,2016; Meitinger et al,2016). Other wellcharacterized centriolar proteins are the components of the centriolar appendages, substructures present only in mature centrioles (i.e., those that are at least two cell cycle old). Centriolar appendages protrudes from the distal side of the centriole usually displaying a nine-fold radial symmetry and they are classified into two types depending on their position within the centriole: the distal and the subdistal appendages (Le Guennec et al,2021). Centriolar proteins required to build these structures have been identified including ODF2, CEP128, Centriolin, NDEL1, Ninein, and CEP170 as 1Departamento de Gen etica, Facultad de Biolog ıa, Universidad de Sevilla, Sevilla, Spain 2Centro Andaluz de Biolog ıa Molecular y Medicina Regenerativa-CABIMER, Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, Sevilla, Spain 3Genome Data Science, Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain 4Department of Cell Biology, Sciences III, University of Geneva, Geneva, Switzerland *Corresponding author. Tel: +34 954 467 667; Fax: +34 954 461 664; E-mail: [email protected] **Corresponding author. Tel: +34 954 467 667; Fax: +34 954 461 664; E-mail: [email protected] ª2023 The Authors. Published under the terms of the CC BY NC ND 4.0license. EMBO reports e56724 |2023 1of 18 key components required to build the subdistal appendages. From a functional point of view, among other roles, distal appendages are essential for cilia assembly and subdistal appendages are involved in MTs anchoring to the centriole (Hall & Hehnly, 2021). Interestingly, it has been proposed that the centrosome plays also cellular functions not directly related to its MTOC activity. One of these functions involves its role as a signaling platform to regulate several cellular processes such as cell polarization, cell cycle regulation or cell proliferation (Arquint et al,2014). Intriguingly, several components of the DNA damage response (DDR) have been identified at the centrosome as the checkpoint proteins ATM, ATR, Chk1, or Chk2 and the repair factors BRCA1, BRCA2, or PARPs (Mullee & Morrison, 2016). Additionally, several centrosomal proteins have been shown to play a direct role in specific DNA damage repair pathways, for example, Centrin-2 in nucleotide excision repair (Dantas et al,2011), or PCNT and CEP164 in checkpoint triggering by ATR and ATM activation (Griffith et al,2008; Sivasubramaniam et al,2008), although the role of CEP164 in DNA repair has been recently disputed (Daly et al,2016). However, it remains unclear if and how the centrosome itself plays a role specifically in the regulation of the DNA double-strand breaks (DSBs) repair pathways. Double-strand breaks are one of the most challenging DNA lesions to be fixed. As the rest of DNA damage, they can arise due to exposure to exogenous (chemicals, irradiation, etc.) or endogenous (transcription or replication problems) (Jackson & Bartek, 2009; Ciccia & Elledge, 2010; Tubbs & Nussenzweig, 2017). In contrast to other DNA lesions, DSBs lack an intact DNA strand to use as a template during repair, hence they are particularly complex to repair. Indeed, DSBs appearance triggers a complex response, the already mentioned DDR, which overhauls the whole cellular metabolism. The DDR is triggered mainly by the activation of the kinases ATM, ATR, Chk1 and Chk2 (Ciccia & Elledge, 2010; Blackford & Jackson, 2017), in a complex process that, as mentioned, requires partially centrosomal components (Mullee & Morrison, 2016). Furthermore, DSBs could be repaired by using one out of several possible mechanisms that can be grouped in two major families: the homologous recombination pathways (HR) or the non-homologous end-joining pathway (NHEJ) (Jasin & Rothstein, 2013; Chang et al,2017; Ranjha et al,2018). Upon the detection of a DSB, the cell needs to commit to one of these two alternative repair pathways, and the outcome of the repair in terms of fidelity is dependent on this decision. The choice is influenced by different cellular cues, such as cell cycle stage. HR can almost only be executed after DNA replication (in S and G2 phases) when the sister chromatid is present and can be used as a template to repair the damaged molecule (Jasin & Rothstein, 2013; Symington, 2016). On the contrary, NHEJ that relies on the ligation of the two ends of DNA with little processing and without a template, can be performed through the whole cell cycle (Chang et al,2017). A complex molecular network is involved in determining the repair pathway of choice (HR vs. NHEJ) upon the detection of a DSB, thus affecting DSB repair outcome. Nevertheless, it happens mostly by controlling the initial step of HR, the so-called DNA end resection (Cejka, 2015; Symington, 2016). This consists in the extensive processing of the DNA ends by nucleases to form long tails of 30OH protruding of single stranded DNA (ssDNA). These ssDNAs are initially coated by the RPA complex (RPA1, RPA2, and RPA3), which protects it from degradation and from pairing with other ssDNAs. Importantly, resected DNA effectively blocks NHEJ, hence committing the repair through HR. Mostly, cellular cues that promote the licensing of DNA end resection, thus promoting HR, affect the activation of the nuclease activity of the MRN complex (MRE11-RAD50-NBS1) through the regulation of CtIP and its partner BRCA1 (Cejka, 2015; Symington, 2016). This triggers the so-called short-range DNA end resection that is followed by a more extensive resection, known as long-range resection (Cejka, 2015; Symington, 2016). From that point onward, HR can happen by different means in a complex molecular choreography that ends with the use of a homologous template for repair (Jasin & Rothstein, 2013). Among other factors, it usually requires the recruitment of the recombinase Rad51 that modulates the invasion of the homologous template. Interestingly, among the proteins identified to localize to the centrosome, in addition of proteins involved in the DDR (such as ATM, ATR, Chk1 or Chk2) there is a subgroup of them linked directly to DSBs repair by HR (i.e., BRCA1 and BRCA2). Thus, we wondered whether the centrosome might play a direct role in HR and, more specifically, in regulating the choice between this repair mechanism and NHEJ by affecting DNA end resection. Strikingly, we observed that cells without centrioles are defective in DNA end resection and HR, supporting this hypothesis. Moreover, such regulation of DNA end resection at centrioles relies on the subdistal appendages. We found that phosphorylation of the subdistal appendage protein CEP170 by the checkpoint kinases ATM and ATR is required to fully activate HR pathway. In the absence of centrioles, subdistal appendages or CEP170, HR is defective, rendering cells hypersensitive to DNA damaging agents. Strikingly, CEP170 levels are altered in several tumors. Interestingly, low levels of the protein increase their mutational burden with specific mutational signatures, chiefly among them the one associated with HR defect. Furthermore, low levels of CEP170 levels correlates with poorer cancer prognosis. Results The centrosome promotes the repair of DNA double-strand breaks through homologous recombination Despite the aforementioned connection between the centrosome and the DDR, it is unknown if there is a direct centrosomal role on DSB repair pathway choice. Intrigued by this possibility, we generated U2OS cells without centrosomes using the PLK4 inhibitor centrinone (Wong et al,2015). As mentioned, PLK4 activity is required for centriole duplication, thus its inhibition leads to cells that while dividing in subsequent cell cycles dilute their preexisting centrioles rendering to the appearance of centrosome-less cells in the population. Centriolar loss requires therefore several cell divisions, hence cannot be observed upon short treatments but only after exposure to the drug for several days (see Fig EV1A and B comparing the 1 vs. 7 days timepoints). We then used this condition to test the impact of centriole loss on the balance between NHEJ and HR repair pathway choice (Fig 1A and B). We used a duo of stably transfected GFP-based repair reporters in which the ectopic expression of the I-SceI nuclease fused to BFP (Blue fluorescent protein) triggers the reconstitution of an active GFP when a specific repair pathway, either HR or NHEJ, is activated (Pierce et al,1999; Bennardo et al,2008) (Fig 1A and B). In this experimental setup, viral transduction efficiency for all 2of 18 EMBO reports e56724 |2023 2023 The Authors EMBO reports Guillermo Rodr ıguez-Real et al 14693178, 0, Downloaded from https://www.embopress.org/doi/10.15252/embr.202256724 by Universidad De Sevilla, Wiley Online Library on [07/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License conditions was monitored and only BFP positive cells were taken into consideration for GFP analysis (for more details, see Materials and Methods). Strikingly, centriole-depleted cells, i.e., treated with centrinone for 7 days, compromised the homologous recombination Rad51-dependent gene conversion pathway (Fig 1A) and at the same time promoted the error prone NHEJ repair (Fig 1B), suggesting that indeed centrosomes are critical to balance DSB-repair pathway choice. As introduced, this decision occurs usually by regulating HR, and mostly by licensing or not DNA end resection. To dissect at which step the centrosome impinges on this process, we monitored A I-SceI target site GFP iGFP iGFP GFP iGFP GFP I-SceI-mediated DSB Gene conversion B GFP puro GFP GFP GFP I-SceI target site total-NHEJ I-SceI-mediated DSBs 4.5 5.0 5.5 6.0 6.5 7.0 DNA content (au) EdU intensity (au) 2n 4n 2n 4n DMSO Centrinone 2% 1%78% 25% 77% 38% F 0 20 40 60 80 100 %Cells >15 RPA foci Centrinone G1 S G2 ns Total G CDE DMSO Centrinone RPA DAPI DMSO Centrinone BRCA1DAPI DMSO Centrinone RAD51DAPI Centrinone Centrinone CentrinoneDMSO DMSO DMSO ______________ _______ G1 phase S phase G2 phase RPA EdU DAPI H DMSO Figure 1. Centrosomes control the balance between repair mechanisms. A, B (A) Homologous recombination was assessed in U2OS cells bearing the DR-GFP reporter in cells lacking centrosomes upon the treatment with centrinone for 7days or mock-treated with DMSO. A schematic representation of the reporter and the recombination event that renders the appearance of a functional GFP geneis shown on the left side. Within the BFP-positive cell population, that is, cells transfected with I-Sce-I containing viral particles, the percentage of GFP-positive cells, that is, cells that have undergone repair by HR after induction with the nuclease I-SceI, was normalized to the DMSO control taken as 1. (B) Same as (A) but in cells harboring the NHEJ reporter EJ5-GFP. C–E(C) U2OS cells treated for 7days with centrinone or DMSO, as indicated, were irradiated with 10 Gy and 1h later prepared for immunofluorescence using an anti- RPA antibody as described in the Materials and Methods section. The number of RPA foci per cell for at least 200 cells per condition was quantified automatically using FIJI software and plotted. Error bars represent standard deviations. One representative experiment out of three performed with similar results is shown. (top). Representative images are shown (bottom). Scale bar in white represents 7lm. (D) Same as (C), but using an antibody against BRCA1. (E) Same as (C), but using an anti-RAD51 antibody. FU2OS cells were treated 7days with centrinone or DMSO as control before a 30-min pulse-labeled with EdU. Afterward, cells were irradiated (10 Gy) and incubated for 1h. Click-it reaction and immunostaining against RPA was carried out as described in the Materials and Methods section. Automated multichannel wide-field microscopy for quantitative image-based cytometry (QIBC) was performed. Single-cell data for more than 5,000 cells is depicted as scatter plots showing DNA content (DAPI intensity) and EdU incorporation during S phase (EdU intensity). Correlatively, RPA foci was measured for each cell. Cells with more than 15 RPA foci are classified as positive (labeled in red). Dotted lines define regions assigned as G1(bottom left), S (top), and G2/M (bottom right). G Quantification of RPA foci positive cells regarding cell cycle status from three different experiments analyzed as described in (F). Each color datapoints marks a biological replica of a total of three independent repetitions. H Representative images of (F) and (G) are shown. Scale bar in white represents 7lm. Data information: In (A, B, and G) the average and standard deviation of three independent experiments is shown. The statistical significance was calculated using a Student’st-test. P-values are represented with one (P<0.05), three (P<0.001) or four (P<0.0001) asterisks. Non-statistical significance is labeled ns. Source data are available online for this figure. 2023 The Authors EMBO reports e56724 |2023 3of 18 Guillermo Rodr ıguez-Real et al EMBO reports 14693178, 0, Downloaded from https://www.embopress.org/doi/10.15252/embr.202256724 by Universidad De Sevilla, Wiley Online Library on [07/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License by immunofluorescence key steps of the homologous recombination repair pathway. Specifically, we inspected the steps of DNA end resection (RPA foci), of HR initiation/commitment (BRCA1 foci), and strand invasion for HR (RAD51 foci) in U2OS cells depleted for centrioles and challenged with 10 Gy of ionizing radiation. Centrosomedepleted cells showed a significant drop in all three steps of HR (Fig 1C–E) suggesting that centrosomes are required to activate HR from the earliest step, i.e., affecting already the licensing of DNA end resection. Similar resection impairment was observed in centrinone treated U2OS cells when DNA damage was induced by Camptothecin (Fig EV1C) or in centrinone treated RPE-1 cells irradiated with 10 Gy (Fig EV1D). To discard that centrinone itself could directly have an effect in HR independently of its effect on centriole duplication, we repeated the RPA foci formation experiment in cells treated with centrinone but only for 1 day, a condition in which the majority of cells retain centrioles, and no impact on resection was observed (Fig EV1E). Finally, to confirm that centriole loss and not extended centrinone treatment by other means impair HR, we repeated the same experimental strategy making use of the HsSAS-6 knock out RPE-1 p53 / cell line. HsSAS-6 is an essential protein for centriole duplication and therefore HsSAS-6 KO cells are devoid of centrosomes and they are only viable in the absence of p53 (Leidel et al,2005; Wang et al,2015). In agreement with our previous results, HsSAS-6 KO p53 / cells also showed a significant decrease in resection compared to control RPE-1 p53 / cells (Fig EV1F). Since all our data supported a role of the centrosome as a regulator of HR, we wondered whether this regulation relied on its presence/absence or the number of centrosomes. Thus, we wondered if cells bearing an excess number of centrosomes could also display an imbalance in HR versus NHEJ. To test this idea, we transiently overexpressed an mCherry-tagged version of PLK4 (Moyer et al,2015) which leads to ~35% of cells bearing extra centrosomes in our cell population (Fig EV1G). Notably, we could not observe any imbalance between HR versus NHEJ in this condition (Fig EV1H and I) suggesting that presence or absence, but not number of centrosomes regulates double-strand break repair pathway choice. We wondered if centrosome loss itself could, somehow, induce the formation of DNA double-strand breaks. To test this idea, we used the phosphorylated form of the histone variant H2AX (cH2AX) as a marker of DSB. As expected, we did not observe any impact on cH2AX foci formation upon centrinone treatment (Fig EV1J). We then checked whether the imbalance observed in centrosomedepleted cells between the two main double-strand break repair pathways would eventually lead to a delay or a lack of repair or if it will simply result on a switch between HR and NHEJ. To do so, we compared the foci dynamics of cH2AX for 6 h after irradiation in control and centriole-depleted cells (Fig EV1K). Notably, we did not observe any significant change in the dynamics of chromosome break repair in centriole-depleted cells, supporting the idea that the decrease of double-strand break repair by HR is countervailed by an increase in NHEJ repair pathway (Fig 1A and B). Intriguingly, we did not observe an overall increase on NHEJ regulators, such as RIF1 nuclear foci, in irradiated centriole-depleted cells (Fig EV1L). If any, we observed a slight reduction on RIF1 accumulation. As RIF1 is an accessory NHEJ factor involved in counteracting resection, our data argue that the increase in NHEJ observed in acentriolar cells does not simply rely on hampering DNA end processing. We then wondered about the mechanism by which the centrosome could be regulating HR versus NHEJ repair pathway choice. We hypothesized that the role of the centrosome in DSB repair might rely on its activity as a microtubule nucleator during interphase. To test this, we measured resection in irradiated U2OS cells pretreated either with MTs depolymerizing (Nocodazole) or with stabilizing (Taxol) drugs (Fig EV1M). Neither of the two treatments mimicked the impact of centrosome loss, suggesting that the connection of the centrosome with DSB repair is independent of its MTOC activity. Overall, we concluded that by a so far uncharacterized mechanism, the centrosome promotes DNA DSB repair by stimulating homologous recombination and impairing NHEJ in a fashion that is independent of its MT organization function. DNA double-strand break repair imbalance upon centrosome loss is not a consequence of cell cycle perturbance Notably, centriole loss through long centrinone treatment has been reported to prolong mitosis. These extended mitosis trigger the activation of 53BP1 and USP28 which eventually blocks the cell cycle in G1 through a p53- and p21-dependent pathway (Wong et al,2015; Fong et al,2016; Lambrus et al,2016; Meitinger et al,2016; preprint: Meitinger et al,2022). As cell cycle is a major regulator of DNA end resection and HR (Huertas, 2010), we wondered if such response could account for the imbalance observed in DNA double-strand break repair. Interestingly, in our experimental conditions, we did not observe either a significant accumulation in G1, an increase of mitotic cells or cell death in cells treated with centrinone (Fig EV1N–Q). Moreover, our results in HsSAS-6 KO cells were already obtained in a p53 KO background, reinforcing the idea that this effect was cell cycle independent (Fig EV1F). Despite that, to further rule out that the absence of centrosomes could be modulating HR indirectly through changes in cell cycle progression mediated by p53, we performed similar experiments in not only Saos-2 cells, a primary osteosarcoma p53 / cell line, but also in RPE-1 lacking either p53 or p21 (Hauge et al,2019). We found a similar decrease in RPA, BRCA1 and RAD51 foci formation to the ones observed in U2OS and RPE-1 cells with all cell lines (Fig EV1R–Z), supporting the idea that the effect of centriolar loss on HR is independent of an accumulation in G1 or mitosis. However, HR takes place during S and G2, and centrinone treatment increases the number of cells in G2 (Fig EV1N). Although we already observed that treatment with camptothecin, that specifically creates DSBs at S phase, was compromised upon centrinone treatment (Fig EV1C), we tested if centriole loss affected equally DNA end resection in these both phases of the cell cycle by using quantitative imagebased cytometry (QIBC) upon irradiation (Fig 1F–H). With this approach, we measured DNA content (DAPI intensity), active DNA replication (with an EdU pulse) and DNA resection (through RPA foci), by fluorescent microscopy (Fig 1F–H). As expected, no resection was observed in G1 cells in any condition. Furthermore, this analysis confirmed that the decrease in resection upon centrinone treatment is similarinbothSandG2phases. Centriolar subdistal appendages proteins promote DNA end resection after double-strand break As stated in the introduction, a few DDR and HR proteins are located at the centrosome (ATM, ATR, CHK1, CHK2, BRCA1, and 4of 18 EMBO reports e56724 |2023 2023 The Authors EMBO reports Guillermo Rodr ıguez-Real et al 14693178, 0, Downloaded from https://www.embopress.org/doi/10.15252/embr.202256724 by Universidad De Sevilla, Wiley Online Library on [07/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License BRCA2) and some centriolar proteins have been previously involved in DDR (Centrin2, CEP63, and PCNT) (Alderton et al,2006; Sivasubramaniam et al,2008; Dantas et al,2011; Mullee & Morrison, 2016), suggesting a close crosstalk between them. We envisaged that the regulation of DSB repair pathway choice by centrosomes will require one or several proteins sharing either of these characteristics: a DDR protein that resides temporarily at the centrosome or a centrosomal-factor that regulates and/or is regulated by the DDR. To search for these factors with an unbiased approach we decided to retrieve previously published data from several publications and databases of either DNA repair/DDR/DDR-regulated factors or centriole/centrosome associated proteins and performed an in silico meta-analysis to look for common factors within these two categories. This meta-analysis retrieved a list of genes previously connected to DDR (1,457 genes) and to centrosome biology (1,424 genes). The crosschecking of these two lists led to the identification of 159 genes linked to both processes (Figs 2A and EV2A; Table EV1). Among these 159 genes, caught our attention a group of three genes previously reported to be part of a very defined substructure of the centrioles, the subdistal appendages (CEP128, NIN, and CEP170) (Hall & Hehnly, 2021). A handful of proteins have been identified to be part of such structure: ODF2, CEP128, Centriolin, NDEL1, Ninein, and CEP170. Their architecture follows a hierarchical organization with ODF2 at the root, and the most proximal to the centriole wall. From that point the structure branches, with a short bough formed by NDEL1 and a second longer one formed by Cep128–Centriolin–Ninein and CEP170, in that order from the inner part to the periphery (Fig 2B). To assess whether the subdistal appendages as a structure is involved in DSB repair, and most specifically in activating HR by licensing DNA end resection, we depleted the above-mentioned proteins using siRNA against them. Depletion of these components was confirmed either by western blot or by RT-PCR, depending on the availability of specific antibodies (Fig EV2B–D). Interestingly, while reduction of ODF2, Centriolin, Ninein and CEP170 showed a significant drop in DNA end resection after DNA damage, albeit to different extents, depletion of NDEL1 did not have a significant impact on the process (Fig 2C). Knockdown of the core resection factor CtIP was used as a positive control. Thus, we conclude that the subdistal appendages, at least the longer branch, is required for fully activation of DNA end resection. Intrigued by these results, we reasoned that the most peripheral component of the long branch of the subdistal appendages, CEP170, could be the critical factor involved in DNA end processing. Therefore, the effect of the depletion of other subdistal appendages proteins might reflect their impact on CEP170 localization to this centriolar structure. To test this idea, we quantified CEP170 levels at the centrosome after depletion of all the subdistal appendages proteins using the same samples that were used for RPA foci formation (Fig 2D). As published, depletion of ODF2, CEP128, Centriolin and Ninein, but not NDEL1, affected CEP170 localization at centrioles to different extents. As expected, downregulation of the core resection factor CtIP did not affect this localization either. Strikingly, and in agreement with our hypothesis, we found a correlation between the decrease of CEP170 levels at the centrosome and the drop on DNA end resection (Fig 2E; R 2 =0.9799). Furthermore, this drop in DNA end resection was also confirmed in the RPE-1 CEP128 KO cell line (Mo ¨nnich et al,2018) where CEP170 levels at the centrosome is also partially reduced (Fig EV2E and F). CEP170 depletion phenotype was rescued by a siRNA-resistant version of the gene (Figs 2F and EV2G). Moreover, the drop in DNA end resection after CEP170 depletion was also observed when cells were challenged with the DNA-damaging agent Camptothecin (Fig EV2H). We also confirmed that CEP170 depletion itself has no impact on RPA or cH2AX foci formation in the absence of an exogenous source DNA damage (Fig EV2I and J). Lastly, we used CRISPR/Cas9 technique to create a KO of CEP170 in RPE cells. Unfortunately, we were not able to recover a full KO, suggesting the protein is essential. However, we created several independent heterozygous CEP170 +/ modified cell lines (Fig EV2K). All those heterozygous cell lines for CEP170 also displayed a clear drop in DNA end resection (Fig 2G). All together, these results revealed that the subdistal appendages are key structures within the centriole involved in DNA end resection and point to CEP170 as the critical mediator of this process. CEP170 promotes homologous recombination from the centrosome Our data suggest that the subdistal appendage component CEP170 promotes DNA end resection after DSB damage, and this DNA processing requires the presence of centrioles. To link those two observations, and test if CEP170 was affecting resection from the centrosome and not due to a putative, unknown, centrosomeindependent function, we tested the effect of CEP170 depletion on DNA end resection in cells without centrioles. As control, we performed similar experiments depleting the HR regulator CtIP. Interestingly, we observed that while CtIP depletion in cells without centrioles further reduced DNA end resection, depletion of CEP170 in cells without centrioles did not show any impact (Fig 3A), thus suggesting that the loss of centrioles is epistatic over CEP170 role but not over a core resection protein such as CtIP. Therefore, the lack of an additive phenotype in centriole-less cells depleted of CEP170 strongly support that CEP170’s role in DDR is played from the centrosome. Furthermore, as the absence of centrioles and the depletion of CEP170 rendered similar resection defect, our data also favor that CEP170 might be the main centrosomal factor accounting for the centrosomes’ role in resection. We then considered that even if CEP170’s role in DDR might require, initially, a centrosomal localization, a possible scenario could be that CEP170 might delocalize to DNA after damage and play a direct role in HR within the nucleus or even at sites of DNA damage. However, an ectopically expressed version of CEP170 tagged with GFP neither relocalized to the nucleus nor was recruited to laser micro-irradiated DNA damage (Fig EV3A). Furthermore, CEP170 protein distribution nucleus versus cytoplasm was also not altered upon irradiation (Fig EV3B), supporting an indirect role of CEP170 in HR repair pathway from the centrosome. So far, we have shown that the centrosome is required for a proper balance between HR and NHEJ, affecting both repair pathways, and that it specifically regulates the initial step of HR, DNA end resection. Also, that the subdistal centriolar protein CEP170 plays a critical role in DNA processing activation. To study whether the role of CEP170, as this of centrosomes, extend not only to DNA end resection but also to both HR and NHEJ, we analyzed the ability of CEP170-depleted cells to perform different types of DSB repair using the GFP-based reporters described above. In agreement with a 2023 The Authors EMBO reports e56724 |2023 5of 18 Guillermo Rodr ıguez-Real et al EMBO reports 14693178, 0, Downloaded from https://www.embopress.org/doi/10.15252/embr.202256724 by Universidad De Sevilla, Wiley Online Library on [07/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License role in DNA end resection, homologous recombination Rad51-dependent gene conversion pathway was compromised (Fig 3B). On the other hand, and in stark contrast with our observation in centrosome-less cells, no significant impact on the NHEJ repair pathway was observed (Fig 3C). In addition to the above-mentioned drop of RPA foci in CEP170-depleted cells after BA E C siNT siCEP170 siNT siCEP170 RPA DAPI GFPGFP-CEP170 siNT siCEP170 GFP GFP-CEP170 siNT siCEP170 F siCtIP siCEP170 siNIN siCNTRL siCEP128 siODF2 siNT siNDEL1 RPADAPI RPADAPI D siCEP170 siNIN siCNTRL siCEP128 siODF2 siNT siNDEL1 CEP170 γ-tubulin/DAPI Control#8#53#60#70 RPA DAPI G CEP170 γ-tubulin Figure 2. CEP170 and the Subdistal appendages regulate DNA end resection. A Venn diagram showing the overlapping of genes found both in DDR screenings and in centrosome-related screening, as described in the main text and the Materials and Methods section. B Schematics of the centrioles showing the Distal and Subdistal appendages (DA and SDA, respectively). The inset shows the molecular architecture of the SDA. CU2OS cells transfected with siRNAs against the indicated proteins or a siRNA control (siNT) and the number of RPA foci per cell for at least 200 cells per condition was quantified automatically using FIJI software and plotted. One representative experiment out of three performed with similar results is shown (top). Representative images are shown (bottom). Scale bar in white represents 7lm. D The same samples used in (C) were used to immunodetect CEP170 at centrioles. Intensity of individual cells is plotted (top) in cells depleted for the indicated factors. Representative images of each condition are shown on the bottom side. For each condition, insets of centrosomes labeled by either CEP170 or c-tubulin are shown. E Linear correlation between RPA foci and CEP170 intensity. For each condition, the average calculated in (C and D) was used in the plot. The R 2 , linear correlation and mathematical formula are shown. FU2OS cells harboring a plasmid expressing a siRNA-resistant GFP-tagged CEP170 or an empty vector were transfected with siRNAs against CEP170 or a control sequence (siNT). Then, cells were treated with IR and the number of RPA foci per cell for at least 200 cells per condition was quantified automatically using FIJI software and plotted. One representative experiment out of three biological replicates that rendered similar results is shown (top). Representative images are shown (bottom). G Same as (C) but in RPE-1cells with partial knock out of CEP170 (top). The number of RPA foci per cell for at least 200 cells per condition was quantified automatically using FIJI software and plotted. One representative experiment out of three biological replicates that rendered similar results is shown. Representative images are shown (bottom). Data information: In (C, D, F, and G), error bars represent standard deviations, the statistical significance was calculated using a Student’st-test. P-values are represented with two (P<0.01), three (P<0.001) or four (P<0.0001) asterisks. Non-statistical significance is labeled ns. Source data are available online for this figure. 6of 18 EMBO reports e56724 |2023 2023 The Authors EMBO reports Guillermo Rodr ıguez-Real et al 14693178, 0, Downloaded from https://www.embopress.org/doi/10.15252/embr.202256724 by Universidad De Sevilla, Wiley Online Library on [07/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License A E H G D siNT siCEP170 BRCA1 DAPI 12 3 4 5 6 7 8 9 12 3 4 6 7 8 9 5 12 3 4 5 6 7 8 9 1 2 3 4 6 7 8 9 5 U2OS GFP U2OS GFP-CEP170 WT U2OS GFP-CEP170 S637A U2OS GFP-CEP170 S637D TOP VIEWSIDE VIEW CEP170 α/β-Tubulin GFP α/β-Tubulin CEP170 α/β-Tubulin GFP α/β-Tubulin GFP α/β-Tubulin GFP α/β-Tubulin GFP α/β-Tubulin GFP α/β-Tubulin GFP α/β-Tubulin GFP α/β-Tubulin IF CEP170 α/β-Tubulin/GFP α/β-Tubulin/ F C B DMSO Centrinone DMSO Centrinone DMSO Centrinone siNT siCEP170 siCtIP RPA DAPI DMSO Centrinone siNT siCEP170 siCtIP DMSO Centrinone DMSO Centrinone siNT siCEP170 RAD51DAPI siNT siCEP170 RIF1DAPI siNT siCEP170 siNT siCEP170 GFP GFP-CEP170 WT GFP-CEP170 S637A GFP-CEP170 S637D siNT siCEP170 siNT siCEP170 RPA DAPI siNT siCEP170 GFP GFP-CEP170 siNT siCEP170 WT S637A S637D siNT siCEP170 siNT siCEP170 Figure 3. 2023 The Authors EMBO reports e56724 |2023 7of 18 Guillermo Rodr ıguez-Real et al EMBO reports 14693178, 0, Downloaded from https://www.embopress.org/doi/10.15252/embr.202256724 by Universidad De Sevilla, Wiley Online Library on [07/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License irradiation, we also observed a drop of BRCA1 or Rad51 foci (Fig 3D and E) and a small but significant reduction on the recruitment of the NHEJ facilitator RIF1 (Fig 3F) along the same lines to the observed changes upon centrinone treatments. Similar changes were observed in CEP128 KO RPE1 cells (Fig EV3C and D). Altogether, our data support that CEP170 and the SDA play a critical role on the centriole subdistal appendages-based regulation of HR. It also suggests that the increase on NHEJ observed in cells lacking centrosomes must be mediated by an additional, yet undiscovered, centriolar factor. CEP170-S637 phosphorylation modulates DNA end resection We then wondered about the molecular steps that might connect DSBs repair and CEP170’s role in HR. Neither cytoplasmic CEP170 protein levels nor overall CEP170 levels changed after irradiationinduced DNA damage (Fig EV3B and E). Remarkably, CEP170 was previously identified in a large-scale proteomic analysis of proteins phosphorylated in response to DNA damage on a consensus site recognized by ATM and ATR (Matsuoka et al,2007). Specifically, Serine-637 of CEP170 is an evolutionarily conserved site in vertebrates (Fig EV3F) identified as a residue phosphorylated after DNA damage (Matsuoka et al,2007). To assess if this putative phosphorylation site is biologically relevant, we generated mutated versions of CEP170 either mimicking (CEP170-S637D) or disrupting this phosphorylation (CEP170-S637A) (Fig EV3G). We confirmed by Ultrastructure Expansion Microscopy (U-ExM) that both mutated versions of CEP170 tagged to GFP, when expressed ectopically, showed a similar localization to the subdistal appendages and the proximal side of the centriole as the one reported for the endogenous protein or the also ectopically expressed wild-type version of GFP-tagged CEP170 (Fig 3G). Only in a minority of cells, but regardless of the version of the protein expressed, the overexpression of the protein rendered the accumulation at additional centrosomal locations (Fig EV3H). Thus, we concluded that this expression of different CEP170 species can be used as a complementation system for the endogenous protein. Interestingly, expression of CEP170-S637D but not CEP170-S637A rescued CEP170-depletion RPA phenotype (Fig 3H), suggesting that not only the presence of the protein, but its phosphorylation, is required to fully activate resection. We hypothesized then that after irradiation, CEP170 is phosphorylated by ATM/ATR in S637, which is critical for promoting HR as the pathway of choice for DSB repair. As expected, the expression of CEP170-S637D alone was unable to rescue the drop of RPA foci formation observed upon chemical inhibition of ATM/ATR (Fig EV3I), in agreement with the fact that CEP170 is just one of many targets involved in homologous recombination that is phosphorylated by ATM/ATR in response to DNA damage. As our results support that CEP170’s role in HR is centrosomal-dependent (Fig 3A), and ATM/ATR have been reported to localize to the centrosome (Mullee & Morrison, 2016), we hypothesized that such phosphorylation happens at that localization. We then wondered whether once phosphorylated, CEP170 function in HR could be centrosome independent. To assess this possibility, we overexpressed the phosphomimic CEP170-S637D mutant in centriole-depleted cells. Interestingly, CEP170-S637D did not compensate the drop in HR observed in centriole-depleted cells (Fig EV3J). Therefore, we concluded that CEP170 location at the centrosome is most likely required for CEP170 phosphorylation by ATM/ATR and for its subsequent role promoting HR. Centrosome/CEP170 DNA damage response contributes to cell survival after DNA double-strand break The above-mentioned results reveal a new function of the centrosome and the centriolar protein CEP170 in DSB repair. To assess whether this function contributes to cell survival and proliferation after DNA damage, we performed clonogenic assays with RPE-1 cells either absent of centrioles or heterozygous by CRISPR for CEP170 (Fig EV2K). In agreement with our hypothesis, both conditions rendered cells hypersensitive to DSB-inducing agents such as ionizing radiation (IR), the radiomimetic agent Neocarzinostatin (NCS), or both topoisomerase poisons Camptothecin (CPT) and Etoposide (VP-16) (Figs 4A–Hand EV4). These hypersensitivities were of a similar extent to the ones observed for other canonical regulators of HR repair pathway choice such as BRCA1 (Cruz-Garc ıa ◀Figure 3. CEP170 phosphorylation at centrosomes is required for resection. AU2OS cells were treated with centrinone or the vehicle DMSO and transfected with the indicated siRNAs. The number of RPA foci per cell for at least 200 cells per condition was quantified automatically using FIJI software and plotted. One representative experiment out of three performed with similar results is shown (left). Representative images are shown (right). Scale bar in white represents 7lm. Statistical significance was calculated using a two-way ANOVA. B, C (B) HR was studied as indicated in Fig 1Ain cells bearing the DR-GFP reporter depleted for the indicated factors. (C) Same as (B), but using cells harboring the NHEJ reporter EJ5-GFP. D–F(D) The number of BRCA1foci per cell for at least 200 cells transfected with a siRNA against CEP170 or a siRNA control was quantified automatically using FIJI software and plotted. One representative experiment out of three performed with similar results is shown (left). Representative image on the right side. (E) Same as (D) but using an antibody against RAD51 (left). Representative images on the right side. (F) Same as (D) but using an antibody against RIF1(top). Representative images at the bottom. G Ultrastructure expansion microscopy (U-ExM) images of U2OS expressing GFP, GFP-CEP170 WT, GFP-CEP170 S637A, or GFP-CEP170 S637D. Images show one representative top view (top panels) and side view (bottom panels). Cells were co-immunostained against alpha and beta tubulin and endogenous CEP170 or GFP as indicated. Orange numbers label the nine SDAs. Orange arrows point to subdistal appendage localization and white arrows to centriole proximal localization of CEP170 variants. Scale bar 500 nm. H Same as (A) but in cells stably expressing the indicated CEP170 variant tagged with GFP or the control GFP plasmid and transfected with siCEP170 or a control sequence (left). Representative images on the right side. The number of RPA foci per cell for at least 200 cells per condition was quantified automatically using FIJI software and plotted. One representative experiment out of three biological replicates that rendered similar results is shown. Scale bar in white represents 7µm. Data information: In (B and C) the average and standard deviation of three independent experiments is shown. For (A, D–F, and H) error bars represent the standard deviation. (A–F and H) the statistical significance was calculated using a Student’st-test. P-values are represented with one (P<0.05), three (P<0.001), or four (P<0.0001) asterisks. Non-statistical significance is labeled ns. Source data are available online for this figure. 8of 18 EMBO reports e56724 |2023 2023 The Authors EMBO reports Guillermo Rodr ıguez-Real et al 14693178, 0, Downloaded from https://www.embopress.org/doi/10.15252/embr.202256724 by Universidad De Sevilla, Wiley Online Library on [07/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License et al,2014). Interestingly, CEP170 knockdown, albeit causing a similar reduction on RPA foci and HR as centrinone treatment, rendered a stronger hyper-sensitivity to DNA damaging agents, probably reflecting the lack of stimulation of NHEJ in that condition when compared with centrinone treatment. Notably, this hypersensitivity to irradiation was also observed in CEP170 siRNA-depleted cells and could be rescued by expressing the siRNA-resistant version of CEP170 (Fig 4I and J). We concluded that the role of the centrosomes and specifically the centriolar protein CEP170 in HR DNA repair is critical to ensure cell survival and proliferation upon DNA damage. CEP170 in cancer biology and cancer prognosis Strikingly, changes in the overall levels of CEP170, either downregulation or upregulation, can be observed in many tumor samples E A F B G C H D IJ 0 Gy 1 Gy 2 Gy 4 Gy GFP siNT GFP siCEP170 GFP-CEP170 siCEP170 GFP siNT GFP siCEP170 GFP-CEP170 siCEP170 01234 0 50 100 Irradiation (Gy) % Survival ns *** *** Figure 4. Centrosomes or CEP170 loss hyper-sensitize to treatment with DNA damaging agents. A–H(A) RPE-1cells knocked out for p53 and heterozygous for CEP170 (red) or a control RPE-1p53 KO cells (blue) were exposed to the indicated doses of ionizing radiation. Cell survival was analyzed as described in the Materials and Methods section using a clonogenic assay. Statistical significance was calculated using a twoway ANOVA. (B) Same as (A) but treating cells with the indicated doses of NCS. (C) Same as (A) but in cells treated with the indicated doses of CPT. (D) Same as (A) but in cells treated with the indicated doses of VP-16. (E) Same as (A) but in RPE p53 / cells treated with centrinone (red) or DMSO (blue) for 7days prior the treatment with ionizing radiation. (F) Same as (E) but in cells treated with the indicated doses of NCS. (G) Same as (E) but in cells treated with the indicated doses of CPT. (H) Same as (E) but in cells treated with the indicated doses of VP-16. IU2OS cells expressing GFP or GFP-CEP170 and transfected with siRNA against CEP170 or non-target sequence were exposed to ionizing irradiation or mock treated. Cell survival was analyzed as in (A). J Representative scanned images of clonogenic assay represented in (I). Data information: Data shown in all panels are the average of three biological independent experiments each of them performed in three technical replicates. Statistical significance was calculated using a two-way ANOVA Error bars represent standard deviations. P-values are represented with two (P<0.01), three (P<0.001) or four (P<0.0001) asterisks. Non-statistical significance is labeled ns. Source data are available online for this figure. 2023 The Authors EMBO reports e56724 |2023 9of 18 Guillermo Rodr ıguez-Real et al EMBO reports 14693178, 0, Downloaded from https://www.embopress.org/doi/10.15252/embr.202256724 by Universidad De Sevilla, Wiley Online Library on [07/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Tumor mutation calls were retrieved from the PCAWG resource, containing 2,658 whole-cancer genomes and their matching normal tissues across 38 tumor types (Aaltonen et al,2020). To identify mutational signatures, we considered only single nucleotide variants (SNVs). A total of 21,628,933 SNVs were used to generate a matrix count of the SBS96 contexts using the SigProfilerMatrixGenerator software (Bergstrom et al,2019). Mutational signatures and its COSMIC reference similarity were inferred via the non-negative matrix factorization algorithm implemented in the SigProfilerExtractor software (preprint: Islam et al,2020). Finally, we selected those samples that also contained expression data (435), divided them into quartiles of CEP170 expression and plotted mutational signature activities corresponding to the first quartile (Q1; low) and fourth quartile of expression (Q4; high) as boxplots using Rstudio (RStudio, 2022). Statistical analysis Statistical significance was determined with a Student’s t-test or two-way ANOVA test as indicated using PRISM software (Graphpad Software, Inc.), except for mutational signature activities comparisons between low and high CEP170 expression were Wilcoxon test was applied using Rstudio (RStudio Team, 2022). Statistically significant differences were labeled with one, two, three or four asterisks for P<0.05, P<0.01, P<0.001 or P<0.0001, respectively. Data availability Our study includes no data deposited in public repositories. Original microscopy images can be found at the BioImage Archive with the BioStudies accession number S-BIAD821 (https://www.ebi.ac.uk/ biostudies/studies/S-BIAD821). Expanded View for this article is available online. Acknowledgements We thank Maikel Castellano-Pozo and Sonia Jimeno for critical reading of the manuscript. We thank CABIMER microscopy facility and Dr. Paloma Dom ınguez for her technical support in image acquisition and processing. Also, Antonio Araujo Due~ nas for helping with the graphics and designing the cover. This work was funded by the R+D+I grant US-1255532 Proyectos I+D+i FEDER Andaluc ıa2014-2020 from the Junta de Andaluc ıa, the grant P18-RT- 1204 from the Consejer ıa de Transformaci on Econ omica, Industria, Conocimiento y Universidades, Junta de Andaluc ıa and the grant PID2019- 104195G from the Spanish Ministry of Science and Innovation-Agencia Estatal de Investigaci on/10.13039/501100011033. CABIMER is supported by the regional government of Andaluc ıa (Junta de Andaluc ıa). ADC is funded with FPU fellowships from the Spanish Ministry of Education and RPC is funded with an AECC postdoctoral fellowship. We also want to thank Paul Guichard and Virginie Hamel (University of Geneva, Department of Cell Biology, Sciences III, Geneva, Switzerland) for their support developing the U-ExM. Author contributions Guillermo Rodr ıguez-Real: Conceptualization; data curation; formal analysis; investigation; methodology; writing –review and editing. 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